Compositions and methods for detecting Cryptococcus neoformans

ABSTRACT

Disclosed are oligonucleotides useful in methods for determining whether a sample contains  Cryptococcus neoformans , a causative agent for human cryptococcosis. These oligonucleotides, which have nucleotide sequences derived from a coding segment of the gene encoding the fungal specific transcription factor gene in  Cryptococcus neoformans , are useful as forward and reverse primers for a polymerase chain reaction using nucleic acids from a biological sample as templates, and as probes for detecting any resultant amplicon. Detection of an amplicon indicates the sample contains  Cryptococcus neoformans . Real-time PCR and detection using florescence resonance energy transfer is disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a divisional of U.S. Ser. No. 12/214,317,filed Jun. 18, 2008, now U.S. Pat. No. 7,989,186, which claims thebenefit of priority under 35 U.S.C. §119(e) to U.S. ProvisionalApplication No. 60/936,628 filed Jun. 20, 2007, the entire contents ofeach of the above-referenced patent applications are hereby incorporatedby reference in their entireties.

FIELD OF THE INVENTION

Example embodiments are generally directed to sensitive and specific PCRassays for detecting Cryptococcus neoformans. Example embodiments alsoprovide forward and reverse primers to target against the fungalspecific transcription factor gene in Cryptococcus neoformans, as wellas hybridization probes containing a fluorescent moiety, such asfluorescein, so as to render a real-time PCR assay to detect thepresence of Cryptococcus neoformans. Other example embodiments mayinclude assays employing conventional PCR to detect the presence ofCryptococcus neoformans.

BACKGROUND

Cryptococcus neoformans is an encapsulated fungus, and is theetiological agent of human cryptococcosis. This fungal pathogenrepresents an opportunistic organism and mainly infectsimmuno-compromised and immuno-competent individuals. There are roughly0.4-1.3 cases per 100,000 per year in the general population. Among AIDSpatients, however, the annual incidence is 200-700 cases per 100,000.The species Cryptococcus neoformans is composed of three variants;namely Cryptococcus neoformans v. gattii, Cryptococcus neoformans v.grubii, and Cryptococcus neoformans v. neoformans. The grubii andneoformans variants have a worldwide distribution and are often found insoil which has been contaminated by bird excrement. Infections aretypically acquired by inhalation of the desiccated fungal cells known asbasidiospores. Primary infection with Cryptococcus neoformans is thoughtto be common early in life where the disease can either lie dormant inthe lungs or disseminate to the central nervous system. With pulmonarydisease, the infection may be acute or chronic. If the fungal pathogenis able to disseminate, infection can present itself asmeninogencephalitis, cutaneous lesions, and other inflammatory symptoms.

Conventional detection methods for Cryptococcus neoformans includeculture on fungal media, colony morphology and nutritionalcharacteristics. Fungi (such as Cryptococcus neoformans) may take 2-3days or even weeks to grow in culture. This makes timely identificationdifficult and greatly impairs efficient treatment where rapididentification of fungal genus/species is required. Most of thesemethods are either time-consuming, laborious, or provide inconclusiveresults.

U.S. Pat. Nos. 5,464,743, 5,580,971, 5,763,163, 5,763,169, 5,707,802,and 6,180,339 disclose detection of fungus including Cryptococcusneoformans using a PCR assay to detect ribosomal RNA (rRNA). Thedesigned probes are targeted against 28S rRNA. U.S. Pat. No. 6,180,339describes amplification of a 401 bp fragment spanning the large subunitrRNA/intergenic spacer region. This PCR-based assay has limitationbecause of its lack of specificity. Multiple probes may be required todistinguish among fungal species.

U.S. Pat. No. 5,919,617 discloses, besides immunological means, the useof primer pairs to amplify the saccharopine dehydrogenase gene to detectCandida albicans.

SUMMARY

There is a continuing need for a specific, rapid and sensitive PCR assayto detect the presence of Cryptococcus neoformans in a biological samplefrom a patient for early diagnosis and treatment. More specifically,there is a need for a highly specific PCR assay to distinguishCryptococcus neoformans from other microorganisms. Disclosed herein areoligonucleotide primers and hybridization probes for use in polymerasechain reaction (“PCR”) methods, such as real-time PCR methods, forspecifically detecting Cryptococcus neoformans in a patient. Alsodisclosed are sensitive and specific PCR assays for detectingCryptococcus neoformans. Further disclosed are kits that include sucholigonucleotide primers and/or hybridization probes.

Provided herein are isolated oligonucleotide primers having about 15-25nucleotides in length, which are capable of annealing under PCRconditions (such as real-time PCR conditions) to a segment of the fungalspecific transcription factor gene of Cryptococcus neoformans. Thesegment of the fungal specific transcription factor gene may, forexample, have a consensus sequence that has at least a 99%, 95% or 90%identity to the following sequences: (a) nucleotides 1121-1144 of SEQ IDNO: 1, (b) nucleotides 1480-1499 of SEQ ID NO: 1, (c) nucleotides2105-2124 of SEQ ID NO: 1, (d) nucleotides 1512-1533 of SEQ ID NO: 2,(e) nucleotides 1165-1184 of SEQ ID NO: 2, and (f) nucleotides 508-527of SEQ ID NO: 2.

Example embodiments provide an isolated oligonucleotide primerconsisting essentially of nucleotide sequence set forth in SEQ ID NO: 3,the nucleotide sequence set forth in SEQ ID NO: 4, the nucleotidesequence set forth in SEQ ID NO: 6, the nucleotide sequence set forth inSEQ ID NO: 7, the nucleotide sequence set forth in SEQ ID NO: 8, and thenucleotide sequence set forth in SEQ ID NO: 9.

The term “consisting essentially of” throughout this application isintended to encompass sequences having at least 99%, 95% or 90% identityto those identified herein. Thus, the following embodiments are examplesthereof.

Example embodiments provide an isolated oligonucleotide, which has atleast about 99% identity to a nucleotide sequence of (a) nucleotides1121-1144 of SEQ ID NO: 1, (b) nucleotides 1480-1499 of SEQ ID NO: 1,(c) nucleotides 2105-2124 of SEQ ID NO: 1, (d) nucleotides 1512-1533 ofSEQ ID NO: 2, (e) nucleotides 1165-1184 of SEQ ID NO: 2, and (1)nucleotides 508-527 of SEQ ID NO: 2.

Further example embodiments provide an isolated oligonucleotide, whichhas at least about 95% identity to a nucleotide sequence of (a)nucleotides 1121-1144 of SEQ ID NO: 1, (b) nucleotides 1480-1499 of SEQID NO: 1, (c) nucleotides 2105-2124 of SEQ ID NO: 1, (d) nucleotides1512-1533 of SEQ ID NO: 2, (e) nucleotides 1165-1184 of SEQ ID NO: 2,and (f) nucleotides 508-527 of SEQ ID NO: 2.

Further example embodiments provide an isolated oligonucleotide, whichhas at least about 90% identity to a nucleotide sequence of: (a)nucleotides 1121-1144 of SEQ ID NO: 1, (b) nucleotides 1480-1499 of SEQID NO: 1, (c) nucleotides 2105-2124 of SEQ ID NO: 1, (d) nucleotides1512-1533 of SEQ ID NO: 2, (e) nucleotides 1165-1184 of SEQ ID NO: 2,and (f) nucleotides 508-527 of SEQ ID NO: 2.

Other example embodiments provide an isolated hybridization probe. Thehybridization probe has a nucleotide sequence consisting essentially ofa sequence complementary to consensus nucleotide sequence of nucleotides1150-1178 of SEQ ID NO: 1 or nucleotides 1583-1611 of SEQ ID NO: 2. Thehybridization probe may include a fluorescent reporter group, moleculeor moiety, such as a fluorescein moiety, e.g., at its 5′ or 3′ end.According to example embodiments, the fluorescent moiety may be a6-carboxy-fluorescein.

In example embodiments, the hybridization probe may have the nucleotidesequence set forth in SEQ ID NO: 5.

According to example embodiments, the hybridization probe may have atleast about 99%, 95% or 90% identity to a nucleotide sequencecomplementary to consensus nucleotide sequence of nucleotides 1150-1178of SEQ ID NO: 1 or nucleotides 1583-1161 of SEQ ID NO: 2.

According to non-limiting example embodiments, provided are methods ofdetecting the presence of Cryptococcus neoformans in a biologicalsample. Such methods include:

(a) mixing (i) extracted DNA obtained from said biological sample, and(ii) a primer pair containing a forward primer and a reverse primer,which target fungal specific transcription factor gene of Cryptococcusneoformans,

(b) amplifying, in a PCR reaction, under conditions to allow productionof an amplicon; and

(c) detecting the presence or absence of Cryptococcus neoformans, in thesample.

According to further non-limiting example embodiments, methods areprovided for detecting the presence of Cryptococcus neoformans in abiological sample using real-time PCR. Such methods include:

(a) mixing (i) extracted DNA obtained from the biological sample, (ii) aprimer pair containing a forward primer and a reverse primer, thattarget fungal specific transcription factor gene of Cryptococcusneoformans, and (iii) a hybridization probe that targets fungal specifictranscription factor gene of Cryptococcus neoformans in a PCR vessel,wherein the hybridization probe includes a fluorescent moiety;

(b) amplifying, in a real-time PCR reaction, under conditions to allowproduction of an amplicon; and

(c) detecting the presence or absence of Cryptococcus neoformans, bydetecting the presence or absence of a fluorescent signal resulting fromthe formation of the amplicon, wherein the presence of a fluorescentsignal is indicative of the presence of Cryptococcus neoformans.

According to non-limiting example embodiments, the forward primerconsists essentially of nucleotide sequence 1121-1144 set forth in SEQID NO.: 1, nucleotide sequence 1480-1499 set forth in SEQ ID NO: 1, ornucleotide sequence 2105-2124 set forth in SEQ ID NO: 1, the reverseprimer consists essentially of nucleotide sequence 1512-1533 set forthin SEQ ID NO: 2, nucleotide sequence 1165-1184 set forth in SEQ ID NO:2, or nucleotide sequence 508-527 set forth in SEQ ID NO: 2, and thehybridization probe consists essentially of nucleotide sequence1150-1178 of SEQ ID NO: 1 or nucleotide sequence 1583-1611 of SEQ ID NO:2.

According to further non-limiting example embodiments, the forwardprimer has at least 99%, 95% or 90% identity to a nucleotide sequence1121-1144 set forth in SEQ ID NO: 1. nucleotide sequence 1480-1499 setforth in SEQ ID NO.: 1, or nucleotide sequence 2105-2124 in SEQ IDNO: 1. The reverse primer has at least 90%, 95% or 99% identity to anucleotide sequence 1512-1533 set forth in SEQ ID NO: 2, nucleotidesequence 1165-1184 set forth in SEQ ID NO.: 2, or nucleotide sequence508-527 set forth in SEQ ID NO: 2, and the hybridization probe has atleast 90%, 95% or 99% identity to a nucleotide sequence 1150-1178 of SEQID NO: 1 or nucleotide sequence 1583-1611 of SEQ ID NO: 2.

According to example embodiments, the forward primer consistsessentially of a nucleotide sequence set forth in SEQ ID NO: 3, and areverse primer consists essentially of a nucleotide sequence set forthin SEQ ID NO: 4. According to other example embodiments, the forwardprimer consists essentially of a nucleotide sequence set forth in SEQ IDNO: 6, and a reverse primer consists essentially of a nucleotidesequence set forth in SEQ ID NO: 7. According to other exampleembodiments, the forward primer consists essentially of a nucleotidesequence set forth in SEQ ID NO: 8, and a reverse primer consistsessentially of a nucleotide sequence set forth in SEQ ID NO: 9.

According to example embodiments, the fluorescent moiety may be afluorescein, and more particularly, 6-carboxy-fluorescein, which may beattached at a 5′- or 3′ end of the hybridization probe.

In other example embodiments kits are provided for PCR for detection ofCryptococcus neoformans. Example kits may include the following:

-   -   (a) a forward primer that anneals to a Cryptococcus neoformans        consensus sequence consisting essentially of nucleotides        1121-1144 of SEQ ID NO: 1;    -   (b) a reverse primer that anneals to a Cryptococcus neoformans        consensus sequence consisting essentially of nucleotides        1512-1533 of SEQ ID NO: 2; and    -   (c) instructions for using said forward primer and reverse        primer in performing PCR to detect a presence of Cryptococcus        neoformans in a sample.

Other example kits may include the following:

-   -   (a) a forward primer that anneals to a Cryptococcus neoformans        consensus sequence consisting essentially of nucleotides        1480-1499 of SEQ ID NO: 1;    -   (b) a reverse primer that anneals to a Cryptococcus neoformans        consensus sequence consisting essentially of nucleotides        1165-1184 of SEQ ID NO: 2; and    -   (c) instructions for using said forward primer and reverse        primer in performing PCR to detect a presence of Cryptococcus        neoformans in a sample.

Other example kits may include the following:

-   -   (a) a forward primer that anneals to a Cryptococcus neoformans        consensus sequence consisting essentially of nucleotides        2105-2124 of SEQ ID NO: 1;    -   (b) a reverse primer that anneals to a Cryptococcus neoformans        consensus sequence consisting essentially of nucleotides 508-527        of SEQ ID NO: 2; and    -   (c) instructions for using said forward primer and reverse        primer in performing PCR to detect a presence of Cryptococcus        neoformans in a sample.

Kits are also provided for real-time PCR in particular. According tosuch embodiments, the kits above may further include a hybridizationprobe having a nucleotide sequence consisting essentially of a sequenceset forth in SEQ ID NO: 5, wherein the probe includes a fluorescentmoiety; and the instructions are instructions for using the forwardprimer, reverse primer and hybridization probe in performing real-timePCR in detecting a presence of Cryptococcus neoformans in a sample.

Thus, according to example embodiments, kits are provided for real-timePCR for detection of Cryptococcus neoformans, which include thefollowing:

-   -   (a) a forward primer that anneals to a Cryptococcus neoformans        consensus sequence consisting essentially of nucleotides        1121-1144 of SEQ ID NO: 1;    -   (b) a reverse primer that anneals to a Cryptococcus neoformans        consensus sequence consisting essentially of nucleotides        1512-1533 of SEQ ID NO: 2;    -   (c) a hybridization probe having a nucleotide sequence        consisting essentially of a sequence set forth in SEQ ID NO: 5,        wherein the probe has a fluorescent moiety; and    -   (d) instructions for using the forward primer, reverse primer        and hybridization probe in performing real-time PCR in detecting        the presence of Cryptococcus neoformans.

In other example embodiments, kits are provided for real-time PCR fordetection of Cryptococcus neoformans, which include:

-   -   (a) a forward primer that anneals to a Cryptococcus neoformans        consensus sequence consisting essentially of nucleotides        1480-1499 of SEQ ID NO: 1;    -   (b) a reverse primer that anneals to a Cryptococcus neoformans        consensus sequence consisting essentially of nucleotides        1165-1184 of SEQ ID NO: 2;    -   (c) a hybridization probe having a nucleotide sequence        consisting essentially of a sequence set forth in SEQ ID NO: 5,        wherein the oligonucleotide probe has a fluorescent moiety; and    -   (d) instructions for using the forward primer, reverse primer        and hybridization probe in performing real-time PCR in detecting        the presence of Cryptococcus neoformans.

In other example embodiments, kits are provided for real-time PCR fordetection of Cryptococcus neoformans, which include:

-   -   (a) a forward primer that anneals to a Cryptococcus neoformans        consensus sequence consisting essentially of nucleotides        2105-2124 of SEQ ID NO: 1;    -   (b) a reverse primer that anneals to a Cryptococcus neoformans        consensus sequence consisting essentially of nucleotides 508-527        of SEQ ID NO: 2;    -   (c) a hybridization probe having a nucleotide sequence        consisting essentially of a sequence set forth in SEQ ID NO: 5,        wherein the oligonucleotide probe has a fluorescent moiety; and    -   (d) instructions for using the forward primer, reverse primer        and hybridization probe in performing real-time PCR in detecting        the presence of Cryptococcus neoformans.

BRIEF DESCRIPTION OF THE FIGURES

Embodiments of the invention are herein described, by way ofnon-limiting example, with reference to the following accompanyingFigures:

FIG. 1 depicts the nucleotide sequence of the fungal specifictranscription factor gene (the plus strand) from Cryptococcus neoformans(SEQ ID NO: 1). The sequence complementary to the primers (i.e., Cneo F1and Cneo R1) and hybridization probe (i.e., Cneo Probe) used in PCR isbold-faced. The nucleotide sequence of fungal specific transcriptionfactor gene is available from GenBank Accession Number AE017349.

FIG. 2 depicts the linear range of detection of the Cryptococcusneoformans fungal specific transcription factor gene by Real-Time PCR.The pCneoJE plasmid dilutions (in duplicate) are 1×10⁹, 1×10⁸, 1×10⁷,1×10⁶, 1×10⁵, 1×10⁴, 1×10³, and 1×10² copies/reaction. Using 35 cycles,the real-time PCR assay is reproducibly detecting as well as 100copies/reaction.

FIG. 3 depicts cross-reactivity analysis using real-time PCR with theprimer (i.e., Cneo F1 and Cneo R1) in Cryptococcus neoformans.Quantitation data displays the serial dilutions of 1:10, 1:100 and1:1,000 of the Cryptococcus neoformans ATCC positive control. Noamplification is shown in the ten cocktails tested and the content ofeach pathogen cocktail is available in Table 1.

FIG. 4 depicts an ethidium bromide stained DNA gel revealing theamplicon products after conventional PCR amplification of Cryptococcusneoformans fungal specific transcription factor gene using the primersets of SEQ ID NOs: 6 and 7 (Cneo F2 and Cneo R2, respectively) and SEQID NOs: 8 and 9 (Cneo F3 and Cneo R3, respectively).

FIG. 5 depicts the detection of the Cryptococcus neoformans fungalspecific transcription factor gene by Real-Time PCR. Quantitation datadisplayed (from left to right) are the positive control (i.e., extractedDNA of ATCC® Cryptococcus neoformans: 2344) at 1:10, 1:100 and 1:1,000dilutions. Amplification using primer sets #5 and 6 (against theCryptococcus neoformans var. neoformanis strain JEC21 chromosome 2 andCryptococcus neoformans var. grubii strain H99 anti-phagocytic protein1, respectively) fail to detect the presence of Cryptococcus neoformans.

DETAILED DESCRIPTION

The aspects, advantages and/or other features of example embodiments ofthe invention will become apparent in view of the following detaileddescription, taken in conjunction with the accompanying drawings. Itshould be apparent to those skilled in the art that the describedembodiments of the present invention provided herein are merelyexemplary and illustrative and not limiting. Numerous embodiments ofmodifications thereof are contemplated as falling within the scope ofthe present invention and equivalents thereto. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety.

Provided herein are sensitive and specific methods using PCR, such asreal-time PCR, to detect the presence of Cryptococcus neoformans in abiological sample. In accordance with example embodiments primers andprobes are also provided, which may be used to detect Cryptococcusneoformans. Also provided are kits including such primers and/or probes.Embodiments of the present invention provide much-needed increasedsensitivity of real-time PCR and are proven to have high specificity.

In describing example embodiments, specific terminology is employed forthe sake of clarity. However, the embodiments are not intended to belimited to this specific terminology.

As used herein, the term “primer” refers to a nucleotide sequence whichcan be extended by template-directed polymerization. For the purpose ofthis application, the term “nucleotide sequence” is intended to includeDNA or modification thereof.

As used herein, the term “isolated” refers to a nucleic acid separatedfrom its natural source.

As used herein, an “isolated” oligonucleotide refers to anoligonucleotide that is synthesized chemically (not a naturallyoccurring nucleic acid).

As used herein, “a” or “an” may mean one or more. As used herein,“another” may mean at least a second or more.

As indicated above, the term “consisting essentially of,” throughoutthis application is intended to encompass sequences having at least 99%,95% or 90% identity to those identified herein.

As used herein, the term “biological sample” may include but are notlimited to urine, fluid or tissue samples such as blood (e.g., wholeblood, blood serum, etc), bronchioalveolar lavage, nasal swabs,cerebrospinal fluid, synovial fluid, brain and other neurologicaltissues, cardiac tissue, skin, lymph nodes, and the like from a mammalsuch as a human or domestic animal. Extraction of nucleic acids frombiological samples is known to those of skill in the art.

Oligonucleotide Primers of the Invention

A. Oligonucleotide Primer Sets Target Against the Fungal SpecificTranscription Factor Gene of Cryptococcus neoformans

The present inventors discovered, inter alia, a PCR assay to detectspecifically Cryptococcus neoformans by amplifying, for example, aportion of the fungal specific transcription factor gene located inchromosome 9 of Cryptococcus neoformans var. neoformans strain JEC21.The fungal specific transcription factor gene encodes a transcriptionalprotein in Cryptococcus neoformans. The full length nucleotide sequenceof the fungal specific transcription factor gene in Cryptococcusneoformans var. neoformanis strain JEC21 is publicly available.(National Center for biotechnology Information Access No. 57228846, theentire content of which is hereby incorporated by reference). The fungalspecific transcription factor gene contains two polynucleotide strands(i.e., a 5′ to 3′ “plus” strand and a 3′ to 5′ “minus” strand). Thenucleotide sequence of SEQ ID NO: 1 represents the plus strand (See,FIG. 1). The nucleotide sequence of SEQ ID NO: 2 represents the minus(i.e., reverse complementary) strand of the nucleotide sequence of SEQID NO: 1.

In Cryptococcus neoformans, ribosomal RNA (rRNA) has been used in PCRdetection (See, e.g., U.S. Pat. Nos. 5,464,743, 5,580,971, 5,763,163,5,763,169, 5,707,802, and 6,180,339). However, the PCR amplification ofthe rRNA is not specific (i.e., the primers used to detect rRNA inCryptococcus neoformans also detect other microorganisms). Contrary tothe methods using the rRNA, the present inventors discovered that thefungal specific transcription factor gene is highly specific for thedetection of Cryptococcus neoformans.

Example embodiments herein are therefore drawn to isolatedoligonucleotide primers and primer sets, which are capable of annealingunder highly stringent hybridization conditions, including PCRconditions, and real-time PCR conditions, to a segment of the fungaltranscription factor gene of Cryptococcus neoformans. As used herein, aprimer set contains a pair of primers; that is, a forward primer and areverse primer. In an example embodiment, the primer set (i.e., forwardprimer and reverse primer) sufficiently anneals to the fungal specifictranscription factor gene segment during the PCR conditions; such asreal-time PCR conditions.

Highly stringent hybridization conditions include the followingconditions: 6×SSC and 65° C.; highly stringent hybridization conditionsdescribed in Ausubel et al., 2002, Short Protocols in Molecular Biology,5^(th) edition, Volumes 1 and 2, John Wiley & Sons, Inc., Hoboken, N.J.,the entire contents of which are hereby incorporated by reference; andhighly stringent hybridization conditions described in Ausubel et al.,1997, Short Protocols in Molecular Biology, 3^(rd) edition, John Wiley &Sons, Inc., New York, N.Y., the entire contents of which are herebyincorporated by reference.

Example embodiments also relate to labeled nucleic acids that can act asprobes (i.e., hybridization probes) to facilitate the detection of anamplification product of Cryptococcus neoformans, using an isolatedoligonucleotide primer set. According to example embodiments, whenhybridized to the target gene (e.g., fungal specific transcriptionfactor gene), the hybridization probe (which may carry a fluorescentmoiety such as a fluorescein moiety on its 5′ end and a quencher on its3′ end or a fluorescent moiety on its 3′ end and a quencher on its 5′end), is degraded when there is successful amplification initiated bythe forward primer and reverse primer against the target gene.

The design of specific primers and hybridization probes may beperformed, for example, using a computer program such as Beacon Designer4.02 (Build 402003) (PREMIER Biosoft International, Palo Alto, Calif.).Other equivalent computer programs such as OLIGO (Molecular BiologyInsights, Inc., Cascade, Colo.) may also be used. One of ordinary skillin the art would appreciate the various factors in the primer design.The factors may include, but are not limited to, melting temperatures ofthe primer pairs, length of the primers or probes and size of theamplicon products. Embodiments of the present invention are not limitedto the specific primers and hybridization probes explicitly providedherein. For example, other primers that amplify the fungal specifictranscription factor gene of Cryptococcus neoformans may be used inaccordance with the present invention.

As used herein, the term “Cneo primers” refers to oligonucleotideprimers (i.e., a forward primer—Cneo F and a reverse primer—Cneo R) thatanneal specifically to the fungal specific transcription factor gene inCryptococcus neoformans and thus initiate the amplifying process underPCR conditions. A first primer set contains a forward primer (“Cneo F1”)and a reverse primer (“Cneo R1”). A second primer set contains a forwardprimer (“Cneo F2”) and a reverse primer (“Cneo R2”). A third primer setcontains a forward primer (“Cneo F3”) and a reverse primer (“Cneo R3”).As used herein, the term “amplifying” refers to a process ofsynthesizing nucleic acid molecules that are complementary to bothstrands of a template nucleic acid molecule (e.g., fungal specifictranscription factor gene). Amplifying during a PCR reaction typicallyinvolves several steps such as denaturing the template nucleic acid (atan elevated temperature), annealing primers to the template nucleic acid(at a temperature which is below the melting temperature of theprimers), and elongating from the primers to produce an amplicon. It isto be understood that amplification typically requires the substrates(i.e., deoxyribonucleoside triphosphates) and a DNA polymerase enzyme(e.g., Taq DNA polymerase) as well as appropriate buffer and co-factors(e.g., magnesium chloride etc). The optimal concentrations ofamplification substrates, enzyme and co-factors can conveniently bedetermined by one skilled in the art.

B. Forward Primers of the Fungal Specific Transcription Factor ofCryptococcus neoformans

In example embodiments, a plurality of forward primers is provided.Typically, oligonucleotide forward primers are 15-25 nucleotides inlength. Primers useful in such embodiments may include e.g., anoligonucleotide primer capable of annealing within a portion of thefungal specific transcription factor gene in Cryptococcus neoformans,and thus providing a point of initiation in nucleic acid synthesis in aPCR process. A forward primer typically is single-stranded, and isdesigned to anneal to either the plus strand or minus strand of thefungal specific transcription factor gene.

In a first example embodiment, the nucleotide sequence of a forwardprimer (i.e., Cneo F1; sequence consisting essentially of the sequenceset forth in SEQ ID NO: 3) corresponds to the fungal specifictranscription factor gene segment of the plus strand which consistsessentially of the nucleotides 1121 through 1144 of SEQ ID NO: 1 (See,FIG. 1). This particular forward primer is capable of annealing to theminus strand spanning the bases 1617 through 1640.

In a second example embodiment, the nucleotide sequence of anotherforward primer (i.e., Cneo F2; sequence consisting essentially of thesequence set forth in SEQ ID NO: 6) corresponds to the fungal specifictranscription factor gene segment of the plus strand which consistsessentially of the nucleotides 1480 through 1499 of SEQ ID NO: 1. Thisparticular forward primer is capable of annealing to the minus strandspanning the bases 1262 through 1281.

In a third example embodiment, the nucleotide sequence of anotherforward primer (i.e., Cneo F3; sequence consisting essentially of thesequence set forth in SEQ ID NO: 8) corresponds to the fungal specifictranscription factor gene segment of the plus strand which consistsessentially of the nucleotides 2105 through 2124 of SEQ ID NO: 1. Thisparticular forward primer is capable of annealing to the minus strandspanning the bases through 637 through 656.

Accordingly, the forward primers consist of the nucleotide sequence ofSEQ ID NO: 3, SEQ ID NO: 6, or SEQ ID NO: 8 (as well as sequences havingat least about 99%, 95%, or 90% identity thereto, as described furtherbelow).

C. Reverse Primers of the Fungal Specific Transcription Factor Gene ofCryptococcus neoformans

In example embodiments, a plurality of reverse primers is provided. Aswith the forward primers, oligonucleotide reverse primers may be 15-25nucleotides in length. Reverse primers useful in the present inventionmay include oligonucleotide reverse primers, in conjunction with theforward primers, which anneal to a different portion and on a differentstrand of the fungal specific transcription factor gene in Cryptococcusneoformans, thus providing an appropriate amplicon product in PCR.Similar to the forward primers, the reverse primers may besingle-stranded and can anneal to either the plus strand or minus strandof the fungal specific transcription factor gene. When forward primeranneals to the plus strand, reverse primer anneals to the minus strand,and vice versa.

In a first example embodiment, the nucleotide sequence of a reverseprimer (i.e., Cneo R1; sequence consisting essentially of the sequenceset forth in SEQ ID NO: 4) corresponds to the fungal specifictranscription factor gene segment of the minus strand which consistsessentially of the nucleotides 1512 through 1533 of SEQ ID NO: 2 (See,FIG. 1). This particular reverse primer is capable of annealing to theplus strand spanning the bases 1228-1249.

In a second example embodiment, the nucleotide sequence of a reverseprimer (i.e., Cneo R2; sequence consisting essentially of the sequenceset forth in SEQ ID NO: 7) corresponds to the fungal specifictranscription factor gene segment of the minus strand which consistsessentially of the nucleotides 1165 through 1184 of SEQ ID NO: 2. Thisparticular reverse primer is capable of annealing to the plus strandspanning the bases 1577-1596.

In a third example embodiment, the nucleotide sequence of a reverseprimer (i.e., Cneo R3; sequence consisting essentially of the sequenceset forth in SEQ ID NO: 9) corresponds to the fungal specifictranscription factor gene segment of the minus strand which consistsessentially of the nucleotides 508 through 527 of SEQ ID NO: 2. Thisparticular reverse primer is capable of annealing to the plus strandspanning the bases 2234-2253.

Thus, according to example embodiments, the reverse primers consist ofthe nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 9(as well as sequences having at least about 99%, 95%, or 90% identitythereto, as described further below).

Given what is disclosed herein, one of ordinary skill in the art wouldknow how to design and prepare primer sets that are useful in amplifyinga segment of the fungal specific transcription factor gene inCryptococcus neoformans. As indicated above, the primers and primer setsprovided are merely exemplary.

D. Example Primer Sets

The primer sets are used in combination with PCR reagents under reactionconditions to initiate primer extension. A primer set useful in thepresent embodiments, may include a forward primer that anneals to onestrand of the fungal specific transcription factor gene, and a reverseprimer that anneals to the opposing strand of the fungal specifictranscription factor gene. The location between the forward primers andreverse primers may be optimized with a computer program to yield anappropriate amplicon size.

In example embodiments, a first primer set is provided, which includes aforward primer (Cneo F1) having the nucleotide sequence as set forth inSEQ ID NO: 3 and a reverse primer (Cneo R1) having the nucleotidesequence as set forth in SEQ ID NO: 4.

In other example embodiments, a second primer set is provided, whichincludes a forward primer (Cneo F2) having the nucleotide sequence asset forth in SEQ ID NO: 6, and a reverse primer (Cneo R2) having thenucleotide sequence as set forth in SEQ ID NO: 7.

In other example embodiments, a third primer set is provided, whichincludes a forward primer (Cneo F3) having the nucleotide sequence asset forth in SEQ ID NO: 8, and a reverse primer (Cneo R3) having thenucleotide sequence as set forth in SEQ ID NO: 9.

E. Polynucleotide Length of Primers

One of ordinary skill in the art would recognize the nucleotide lengthrequired for the primer set and would be able to conveniently determinethe optimal length of the primers. In example embodiments, the primershave about 15-25 base-pairs (bp) in length. According to further exampleembodiments, the primers have about 20-22 bp nucleotides.

F. Primers with High Percent Sequence Identity to Sufficiently AnnealDuring PCR

It is understood that the nucleotide sequence of the primers that annealto the fungal specific transcription factor gene in Cryptococcusneoformans may somewhat vary without affecting its annealing ability ina PCR reaction. Thus, the present methods, primers, and kits areintended to encompass these slight variations.

In example embodiments, the forward primer is at least about 99%identical to the fungal transcription factor gene segment correspondingto the nucleotide sequence set forth in SEQ ID NO: 3, SEQ ID NO: 6, orSEQ ID NO: 8. According to further examples, the forward primer has atleast about 95% identity to SEQ ID NO: 3, SEQ ID NO: 6 or SEQ ID NO: 8.According to even further examples, the forward primer has at leastabout 90% identity to SEQ ID NO: 3, SEQ ID NO: 6, or SEQ ID NO: 8.

In other example embodiments, the reverse primer is at least about 99%identical to the fungal specific transcription factor gene segmentcorresponding to the nucleotide sequence set forth in SEQ ID NO: 4, orSEQ ID NO: 7, or SEQ ID NO: 9. According to further examples, thereverse primer has at least about 95% identity to SEQ ID NO: 4, or SEQID NO: 7, or SEQ ID NO: 9. According to even further examples, thereverse primer has at least about 90% identity to SEQ ID NO: 4, or SEQID NO: 7, or SEQ ID NO: 9.

Example embodiments further include isolated oligonucleotide primers,wherein the oligonucleotide primers have at least 99%, 95%, or 90%identity to a nucleotide sequence selected from the group consisting of(a) nucleotides 1121-1144 of SEQ ID NO: 1, (b) nucleotides 1480-1499 ofSEQ ID NO: 1, (c) nucleotides 2105-2124 of SEQ ID NO: 1, (d) nucleotides1512-1533 of SEQ ID NO: 2, (e) nucleotides 1165-1184 of SEQ ID NO: 2,and (1) nucleotides 508-527 of SEQ ID NO: 2.

Pairwise nucleotide sequence alignments and determination of percentidentities may be performed using the default parameters of the ClustalV algorithm or the Clustal W algorithm, wherein both algorithms areincorporated into the Power Macintosh MegAlign 6.1 program (DNASTAR,Madison, Wis.). The default parameters for pairwise alignments using theClustal V algorithm are as follows: Ktuple=1, gap penalty=3, window=5,and diagonals=5. The default parameters for pairwise alignments usingthe Clustal W algorithm are as follows: gap penalty=10.00 and gaplength=0.10. The Clustal V algorithm is described in Higgins et al.,1989, Fast and sensitive multiple sequence alignments on amicrocomputer. Computer Applications in the Biosciences 5:151-153, theentire contents of which are hereby incorporated by reference. TheClustal W algorithm is described in Thompson et al., 1994, CLUSTAL W:improving the sensitivity of progressive multiple sequence alignmentthrough sequence weighting, position specific gap penalties and weightmatrix choice. Nucleic Acids Research 22:4673-80, the entire contents ofwhich are hereby incorporated by reference. In other embodiments, theoligonucleotide and the segment of the polynucleotide include the samenumber of nucleotides.

Hybridization Probe of the Fungal Specific Transcription Factor Gene ofCryptococcus neoformans

Example embodiments are also drawn to isolated oligonucleotidehybridization probes capable of hybridizing under highly stringenthybridization conditions to a segment of a polynucleotide directed tothe fungal specific transcription factor gene of Cryptococcusneoformans. In example embodiments, the hybridization probe may includea dual labeled hybridization probe or a molecular beacon.

As described further herein, example embodiments of PCR assays fordetection of Cryptococcus neoformans include conventional PCR assays andreal-time PCR assays. Real-time PCR offers a much-increased sensitivityfor detection, as compared to conventional PCR, making it an idealassay. A PCR assay in conjunction with fluorescence resonance energytransfer (FRET) technique may be employed for real-time PCR. In such anassay, a donor fluorescent moiety and a corresponding acceptorfluorescent moiety are positioned in such a manner that the energytransfer taking place between two fluorescent moieties can be detectedand monitored via a machine. It is understood that the emission spectrumof an acceptor fluorescent moiety overlaps with the excitation spectrumof a donor fluorescent moiety. One of ordinary skill in the art wouldknow how to select a fluorescent donor moiety and its correspondingacceptor moiety in FRET.

According to non-limiting example embodiments, TAQMAN® technology may beused to detect the presence of an amplification product, i.e., to detectthe presence or absence of Cryptococcus neoformans in a real-time PCRassay. Typically, in FRET involving TAQMAN® technology to detect thepresence of an amplification product, a hybridization probe is labeledwith two fluorescent moieties. When a first fluorescent moiety isexcited (e.g., light with specific wavelength), the absorbed energy istransferred to a second fluorescent moiety in accordance with theprinciple of FRET. When the second fluorescent moiety is a quenchermolecule, the energy transferred is absorbed (i.e., quenched) and nodetection of fluorescence emission can be detected. During the annealingstep of a PCR reaction, a hybridization probe (dually labeled with twofluorescent moieties) is annealed to the fungal specific transcriptionfactor gene. If a successful amplification reaction occurs, the Taqpolymerase then degrades (attributed by the 5′ to 3′ exonucleaseactivity) the hybridization probe. The degradation of hybridizationprobe by Taq polymerase allows the first fluorescent moiety to bespatially separated from the second fluorescent moiety; thus permittingthe detection of fluorescence emission (as the quencher fails to absorbthe transferred energy).

Typically, hybridization probes are about 20-35 base pairs in length, soas to sufficiently anneal to the target nucleic acid molecules (i.e.,fungal specific transcription factor gene). The primers may containe.g., 30 nucleotides. As used herein, “Cneo Probe” refers tooligonucleotide probe that anneals specifically to the fungal specifictranscription factor nucleic acid sequences and provide fluorescencesignals during annealing of PCR polymerization.

In accordance with example embodiments, molecular beacon technology inconjunction with PCR may also be used. In this technology, ahybridization probe is also labeled with a first fluorescent moiety anda second fluorescent moiety. Like TAQMAN® technology, the secondfluorescent moiety is generally a quencher. Typically, molecular beacontechnology uses an oligonucleotide as a hybridization probe that permithairpin formation (i.e., the hybridization probe contains nucleotidesequences to form a hairpin). As a result, the two fluorescent moietiespresent on the hybridization probe are in close proximity when insolution (due to hairpin formation). However, if there is a successfulamplification and annealing, the hybridization probe anneals to thetarget nucleic acids and thus destroys the hairpin formation, separatingthe two florescent moieties and allowing the detection of the emissionof fluorescent energy.

According to non-limiting example embodiments, an isolated hybridizationprobe is provided having a nucleotide sequence consisting essentially ofa sequence complementary to consensus nucleotide sequence of nucleotides1150-1178 of SEQ ID NO: 1 or 1583 through 1611 of SEQ ID NO: 2, wherethe hybridization probe includes at least one fluorescent moiety.According to example embodiments, the hybridization probe anneals to thefungal specific transcription factor gene segment consisting essentiallyof nucleotides 1150 through 1178 of SEQ ID NO: 1 (FIG. 1). In otherexample embodiments, the hybridization probe anneals to the fungalspecific transcription factor gene segment consisting essentially ofnucleotides 1583 through 1611 of SEQ ID NO: 2.

According to example embodiments, the hybridization probe includes atleast one fluorescent moiety (or molecule), such as a fluoresceinmoiety. The fluorescent moiety may be located e.g., at the 5′ end of thehybridization probe. Examples of suitable fluorescent moieties that maybe used in accordance with real-time PCR methods would be known to thoseskilled in the art. For example fluorescent reporter or fluorophore(e.g. 6-carboxy-fluorescein, acronym: FAM, or tetrachlorofluorescein,acronym: TET) may be covalently attached to the 5′ end of the probe.According to non-limiting example embodiments, the fluorescent moleculeis a 6-carboxy-fluorescein moiety attached to the 5′ end of thehybridization probe.

In further example embodiments, the hybridization probe may include aquencher. The fluorescent molecule emits its emission light and isquenched by a quencher during a real-time PCR reaction. The quencher maybe attached to the opposite end of the hybridization probe from thefluorescent moiety, e.g., to the 3′end of the hybridization probe.Example quenchers may include e.g., BLACK HOLE QUENCHER® 1 (version 1quencher of common fluorophores that emit light in the range of 430-730nm), BLACK HOLE QUENCHER® 2 (version 2 quencher of common fluorophoresthat emit light in the range of 430-730 nm), IOWA BLACK® FQ (quencher ofcommon fluorophores that emit light in the range of 420-620 nm), IOWABLACK® RQ-sp (quencher of common fluorophores that emit light in therange of 500-700 nm), and the like.

In example embodiments, the hybridization probe consists essentially ofnucleotide sequence of SEQ ID NO: 5, as well as at least one fluorescentmoiety, and may further include a quencher.

It is understood that the nucleotide sequence of the isolatedhybridization probe may also somewhat vary. The present methods, probes,and kits are intended to encompass these variations as well. Inparticular, according to example embodiments, hybridization probes mayhave at least 99%, 95% or 90% identity to a nucleotide sequenceconsisting essentially of SEQ ID NO: 5. Example embodiments also providean isolated hybridization probe, which has at least about 99%, 95% or90% identity to a nucleotide sequence complementary to consensusnucleotide sequence of nucleotides 1150 through 1178 of SEQ ID NO: 1 ornucleotides 1583 through 1611 of SEQ ID NO: 2.

Oligonucleotide Combinations of the Invention

Other embodiments are directed to a composition (e.g., a reactionmixture or a kit) including a first isolated oligonucleotide (e.g., aforward primer) and a second isolated oligonucleotide (e.g., a reverseprimer).

In example embodiments, the composition includes a first forward primerwhich corresponds to (e.g., includes or consists essentially of) thenucleotides 1121 through 1144 of SEQ ID NO: 1 (i.e., SEQ ID NO: 3), anda first reverse primer which corresponds to (e.g., includes or consistsessentially of) the nucleotides 1512 through 1533 of SEQ ID NO: 2 (i.e.,SEQ ID NO: 4). The forward primer of SEQ ID NO: 3 and reverse primer ofSEQ ID NO: 4 are capable of annealing under PCR conditions to the fungalspecific transcription factor gene segment and produce an amplicon.

In other embodiments, the composition includes a second forward primerwhich corresponds to the nucleotides 1480 through 1499 of SEQ ID NO: 1.(i.e., SEQ ID NO: 6) and a second reverse primer which corresponds tothe nucleotides 1165 through 1184 of SEQ ID NO: 2 (i.e., SEQ ID NO: 7).The forward primer of SEQ ID NO: 6 and reverse primer of SEQ ID NO: 7are capable of annealing under PCR conditions to the fungal specifictranscription factor gene segment and produce an amplicon.

In other embodiments, the composition includes a third forward primerwhich corresponds to the nucleotides 2105 through 2124 of SEQ ID NO: 1.(i.e., SEQ ID NO: 8) and a third reverse primer which corresponds to thenucleotides 508 through 527 of SEQ ID NO: 2 (i.e., SEQ ID NO: 9). Theforward primer of SEQ ID NO: 8 and reverse primer of SEQ ID NO: 9 arecapable of annealing under PCR conditions to the fungal specifictranscription factor gene segment and produce an amplicon.

Example embodiments provide a sensitive and specific PCR assay to detectCryptococcus neoformans. In example embodiments, hybridization probesare provided. The hybridization probe is capable of annealing tonucleotide sequences contained in SEQ ID NO: 1 or SEQ ID NO: 2. Thehybridization probe may be about 20-35 nucleotides long. According toexample embodiments, the hybridization probe is about 30 nucleotideslong.

In example embodiments, the hybridization probe comprises the nucleotidesequence of SEQ ID NO: 5.

Methods

The present inventors discovered that the fungal specific transcriptionfactor gene can be used as a diagnostic tool for determining thepresence of Cryptococcus neoformans in biological samples obtained frommammals such as a human subject. Example embodiments pertains to methodsfor determining whether a sample (e.g., a biological sample such asurine) contains Cryptococcus neoformansi, wherein the methods includethe following: (a) mixing DNA extracted from a biological sample with aprimer pair that targets fungal specific transcription factor gene ofCryptococcus neoformans (e.g., where the primer pair may be included ina composition) (b) amplifying, by a polymerase chain reaction, a segmentof the nucleic acid (directed to the fungal specific transcriptionfactor gene in Cryptococcus neoformans) to produce an amplicon, whereinproduction of the amplicon is primed by the forward and reverse primers,and (c) detecting a presence or absence of Cryptococcus neoformans inthe sample if the amplicon is detected. The forward primer may becapable of annealing under PCR conditions to a polynucleotide consistingof the nucleotide sequence of SEQ ID NO: 2, wherein the reverse primeris capable of annealing to a polynucleotide consisting of the nucleotidesequence of SEQ ID NO: 1. As part of the present methods, a vesselcontaining the primer pair and biological vessel may be may be incubatedunder conditions allowing production of the amplicon if the samplecontains Cryptococcus neoformans.

In other example embodiments, the forward primer may be capable ofhybridizing under PCR conditions to a segment of a polynucleotide,wherein the segment consists essentially of nucleotides 1617 through1640 of SEQ ID NO: 2. In another embodiment, the reverse primer may becapable of hybridizing under the same conditions to a segment of apolynucleotide, wherein the segment consists essentially of nucleotides1228 through 1249 of SEQ ID NO: 1.

In another embodiment, the forward primer is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or is 100% identicalto a segment of a polynucleotide based on the Clustal V or W alignmentmethod using the default parameters, wherein the first polynucleotideconsists of the nucleotide sequence of SEQ ID NO: 1. In anotherembodiment, the forward primer and the segment of the polynucleotidecontain the same number of nucleotides. In another embodiment, thereverse primer is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or 99% identical to, or is 100% identical to a segment of apolynucleotide based on the Clustal V or W alignment method using thedefault parameters, wherein the second polynucleotide consists of thenucleotide sequence of SEQ ID NO: 2. In another embodiment, the reverseprimer and the segment of the second polynucleotide contain the samenumber of nucleotides.

In another embodiment, the forward primer is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or is 100% identicalto a segment of a polynucleotide, wherein the segment consists of thereverse complement of nucleotides 508 through 527 of SEQ ID NO:2 basedon the Clustal V or W alignment method using the default parameters. Inanother embodiment, the reverse primer is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to, or is 100% identical to asegment of a polynucleotide, wherein the segment consists of the reversecomplement of nucleotides 2234 through 2253 of SEQ ID NO: 1 based on theClustal V or W alignment method using the default parameters.

In another embodiment, the forward primer consists of a nucleotidesequence comprised by the nucleotide sequence of SEQ ID NO:1. In anotherembodiment the reverse primer consists of a nucleotide sequencecomprised by the nucleotide sequence of SEQ ID NO:2. In anotherembodiment, the forward or forward primers comprise 15, 16, 17, 18, 19,20, 21, 22, 23, 24, or 25 nucleotides. In another embodiment, theforward or reverse primers are 20-25 nucleotides long. In anotherembodiment, the forward primer includes a sequence consistingessentially of the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 6, orSEQ ID NO: 8. In another embodiment, the reverse primer includes asequence consisting essentially of the nucleotide sequence of SEQ ID NO:4, SEQ ID NO: 7 or SEQ ID NO: 9.

Optionally, the mixing may include mixing a hybridization probe (e.g., ahybridization probe labeled with two fluorescent moieties in accordancewith TAQMAN® technology or a molecular beacon) capable of detecting theamplicon if the amplicon is produced. In other embodiments, theoligonucleotide probe is capable of hybridizing under PCR conditions toa polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1or SEQ ID NO: 2. In other embodiments, the oligonucleotide probe iscapable of hybridizing under PCR conditions to a segment of apolynucleotide, wherein the segment consists essentially of nucleotides1583 through 1611 of SEQ ID NO:2 or nucleotides 1150 through 1178 of SEQID NO:1.

In another embodiment, the hybridization probe is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or is 100%identical to a segment of a polynucleotide based on the Clustal V or Walignment method using the default parameters, wherein thepolynucleotide consists essentially of the nucleotide sequence of SEQ IDNO: 1 or SEQ ID NO: 2. In another embodiment, the oligonucleotide probeand the segment of the polynucleotide contain the same number ofnucleotides.

In another embodiment, the oligonucleotide probe is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or is 100%identical to a segment of a polynucleotide, wherein the segment consistsessentially of the reverse complement of nucleotides 1583 through 1611of SEQ ID NO: 2 or nucleotides 1150 through 1178 of SEQ ID NO: 1 basedon the Clustal V or W alignment method using the default parameters.

In other example embodiments, the oligonucleotide probe includes anucleotide sequence comprised by the nucleotide sequence of SEQ ID NO: 1or SEQ ID NO: 2. In another embodiment, the hybridization probecomprises 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or35 nucleotides. In another embodiment, the oligonucleotide probe is 30nucleotides long. In another embodiment, the oligonucleotide probeconsists essentially of the nucleotide sequence of SEQ ID NO: 5.

In other example embodiments, a 6-carboxy-fluorescein moiety is attachedto the 5′ end of the oligonucleotide probe. In another embodiment, aBLACK HOLE QUENCHER® 1 (version 1 quencher of common fluorophores thatemit light in the range of 430-730 nm) moiety is attached to the 3′ endof the oligonucleotide probe. In other embodiments, the amplicon isdetected by the oligonucleotide probe during real-time PCR. Ifconvenient PCR is used, the formation amplicon is detected by gelelectrophoresis after the PCR is completed.

Non-limiting example embodiments include methods of detecting presenceof Cryptococcus neoformans in a biological sample, that include:

-   -   (a) mixing        -   (i) DNA extracted from the biological sample,        -   (ii) a primer pair comprising a forward primer and a reverse            primer that target a fungal specific transcription factor            gene of Cryptococcus neoformans, and        -   (iii) a hybridization probe, in a PCR vessel, wherein the            hybridization probe comprises a fluorescent moiety;    -   (b) amplifying, in a real-time PCR reaction, under conditions to        allow production of an amplicon; and    -   (c) detecting a presence or absence of Cryptococcus neoformans,        by detecting a presence or absence of a fluorescent signal        resulting from the formation of the amplicon, wherein the        presence of a fluorescent signal is indicative of the presence        of Cryptococcus neoformans.

The primers, probes, fluorescent moieties and other aspects of these andother embodiments may be as described herein throughout.

PCR Detection Assay

Example embodiments provide utilizing amplification approaches toquickly determine the presence of a particular gene. For purposes ofthis application, “amplifying” refers to a process of synthesizingnucleic acid molecules that are complementary to one or both strands ofa template nucleic acid. With respect to Cryptococcus neoformans, thepresent inventors discovered amplifying the fungal specifictranscription factor gene to be a highly sensitive and specific approachto determine the presence of such microorganism. One would appreciatethe extraction protocol by which DNA of fungi can be extracted. Forexample, DNA may be extracted from biological samples using the acidguanidinium thiocyanate-phenol-chloroform method (Chomczymski andSacchi, Analytical Biochemistry, vol. 162, pp. 156-159, 1987). Also, oneof ordinary skill in the art would appreciate that amplifying a nucleicacid molecule typically includes (i) denaturing the template nucleicacid, (ii) annealing primers to the template nucleic acid at atemperature that is below the melting temperatures of the primers, and(iii) enzymatically elongating from the primers to generate anamplification product. Although the denaturing, annealing and elongatingsteps can be performed once, they are generally performed multipletimes. As the amount of amplification product increases (often timesexponentially), it increases the sensitivity of the assay. Typically,amplification requires the presence of a Taq DNA polymerase, anappropriate buffer and appropriate salts such as magnesium chloride orpotassium chloride.

In example embodiments a real-time PCR assay is provided. The real-timePCR assay is more sensitive than a conventional PCR assay, (butconventional PCR assays may be alternatively utilized herewith). Aidedwith the help of DNA probe, the real-time PCR provides a quantum leap asa result of real-time detection. In real-time PCR assay, a fluorometerand a thermal cycler for the detection of fluorescence during thecycling process is used. A computer that communicates with the real-timemachine collects fluorescence data. This data is displaced in agraphical format through software developed for real-time analysis.

In addition to the forward primer and reverse primer (directed to thegene of interest—the fungal specific transcription factor gene), asingle-stranded hybridization probe is also used. The hybridizationprobe may be a short oligonucleotide, usually 20-35 bp in length, and islabeled with a fluorescent reporting dye attached to its 5′-end as wellas a quencher molecule attached to its 3′-end. When a first fluorescentmoiety is excited with light of a suitable wavelength, the absorbedenergy is transferred to a second fluorescent moiety (i.e., quenchermolecule) according to the principles of FRET. Because the probe is only20-35 bp long, the reporter dye and quencher are in close proximity toeach other and little fluorescence is detected. During the annealingstep of the PCR reaction, the labeled hybridization probe binds to thetarget DNA (i.e., the amplification product). At the same time, Taq DNApolymerase extends from each primer. Because of its 5′ to 3′ exonucleaseactivity, the DNA polymerase cleaves the downstream hybridization probeduring the subsequent elongation phase. As a result, the excitedfluorescent moiety and the quencher moiety become spatially separatedfrom one another. As a consequence, upon excitation of the firstfluorescent moiety in the absence of the quencher, the fluorescenceemission from the first fluorescent moiety can be detected. By way ofexample, a Rotor-Gene System is used and is suitable for performing themethods described herein for detecting fungal specific transcriptionfactor gene in Cryptococcus neoformans. Further information on PCRamplification and detection using a Rotor-Gene can conveniently be foundon Corbett's website.

In alternative embodiments, suitable hybridization probes such asintercalating dye (e.g., Sybr-Green I) or molecular beacon probes can beused. Intercalating dyes can bind to the minor grove of DNA and yieldfluorescence upon binding to double-strand DNA. Molecular beacon probesare based on a hairpin structure design with a reporter fluorescent dyeon one end and a quencher molecule on the other. The hairpin structurecauses the molecular beacon probe to fold when not hybridized. Thisbrings the reporter and quencher molecules in close proximity with nofluorescence emitted. When the molecular beacon probe hybridizes to thetemplate DNA, the hairpin structure is broken and the reporter dye is nolong quenched and the real-time instrument detects fluorescence.

The range of the primer concentration can optimally be determined. Theoptimization involves performing a dilution series of the primer with afixed amount of DNA template. The primer concentration may be betweenabout 50 nM to 300 nM. An optimal primer concentration for a givenreaction with a DNA template should result in a low Ct-(thresholdconcentration) value with a high increase in fluorescence (5 to 50times) while the reaction without DNA template should give a highCt-value.

Real-time PCR methods include performing at least one cycling step thatincludes amplifying and hybridizing. An amplification step includescontacting the biological sample (suspected of containing DNA ofCryptococcus neoformans) with a pair of Cneo-primers to produce a Cneoamplification product if a fungal specific transcription factor nucleicacid molecule is present in the biological sample. The forward andreverse primers anneal to a target within or adjacent to a fungalspecific transcription factor nucleic acid molecule such that at least aportion of the amplification product contains nucleic acid sequencecorresponding to fungal specific transcription factor and, moreimportantly, such that the amplification product contains the nucleicacid sequences that are complementary to fungal specific transcriptionfactor gene probes. A hybridizing step also includes contacting thebiological sample with a hybridization probe. The hybridization probe iscomplementary to either the 5′ strand or 3′ strand of the fungalspecific transcription factor gene. According to example embodiments,the hybridization probe anneals to the fungal specific transcriptionfactor gene. When the Cneo primers begin to anneal, the Taq polymerasebreaks the hybridization probe and release donor fluorescent moiety aswell as a corresponding acceptor fluorescent moiety. The method furtherincludes detecting the presence or absence of FRET between the donorfluorescent moiety of the hybridization probe and the correspondingacceptor fluorescent moiety of the hybridization probe. Multiple cyclingsteps can be performed, e.g., in a thermocycler. The above-describedmethods for detecting fungal specific transcription factor gene gene ina biological sample using primers and probes directed toward fungalspecific transcription factor gene also can be performed using otherfungal specific transcription factor gene-specific primers and probes.

Within each thermocycler run, control samples can be cycled as well.Positive control samples can also amplify, for example, a plasmidconstruct containing Cryptococcus neoformans Cneo nucleic acidmolecules. Such a plasmid control can be amplified internally (e.g.,within each biological sample) or in separate samples run side-by-sidewith the patients' samples. Each thermocycler run also should include anegative control that, for example, lacks Cryptococcus neoformans Cneotemplate DNA. Such controls are indicators of the success or failure ofthe amplification, hybridization, and/or FRET reaction. Therefore,control reactions can readily determine, for example, the ability ofprimers to anneal with sequence-specificity and to initiate elongation,as well as the ability of probes to hybridize with sequence-specificityand for FRET to occur.

Real-Time PCR Detection Kit for Cryptococcus neoformans

Example embodiments provide kits of manufacture, which may be used todetect specifically Cryptococcus neoformans. An article of manufacture(i.e., kit) according to the present invention includes a set of primers(i.e., a forward primer and a reverse primer) (directed to fungalspecific transcription factor gene) and optionally a hybridization probe(directed to fungal specific transcription factor gene) used to detectCryptococcus neoformans, contained within a suitable packaging material.Representative primers and hybridization probes provided in the kit fordetection of Cryptococcus neoformans can be complementary to the fungalspecific transcription factor gene encoding an outer surface membraneprotein for Cryptococcus neoformans.

In embodiments including a hybridization probe, the hybridization probemay be conveniently labeled with a fluorescent moiety e.g., at its5′-end and a quencher moiety at its 3′-end. Examples of suitable FRETdonor fluorescent moieties and acceptor fluorescent moieties areprovided herein. Alternatively, the hybridization probes supplied withthe kit can be labeled. For example, an article of manufacture mayinclude an instruction to tag the hybridization probe with a donorfluorescent moiety at its 5′-end and a corresponding acceptorfluorescent moiety at its 3′-end.

Methods of designing primers and hybridization probes are disclosedherein, and representative examples of primers and hybridization probesthat amplify and hybridize to fungal specific transcription factornucleic acids encoding a transcription protein are provided.

Articles of manufacture or kits provided herein may also includeinstructions, such as a package insert having instructions thereon, forusing the primers to detect of the presence of Cryptococcus neoformansin a biological sample. Such instructions may be for using the primerpairs and/or the hybridization probes to specifically detectCryptococcus neoformans in a biological sample.

Non-limiting example embodiments may include kits for PCR detection ofCryptococcus neoformans, which include the following:

-   -   (a) a forward primer that anneals to a Cryptococcus neoformans        consensus sequence consisting essentially of nucleotides        1121-1144 of SEQ ID NO: 1;    -   (b) a reverse primer that anneals to a Cryptococcus neoformans        consensus sequence consisting essentially of nucleotides        1512-1533 of SEQ ID NO: 2; and    -   (c) instructions for using the forward primer and reverse primer        in performing PCR to detect a presence of Cryptococcus        neoformans in a sample.

Other non-limiting example embodiments may include kits for PCRdetection of Cryptococcus neoformans, which include:

-   -   (a) a forward primer that anneals to a Cryptococcus neoformans        consensus sequence consisting essentially of nucleotides        1480-1499 of SEQ ID NO: 1;    -   (b) a reverse primer that anneals to a Cryptococcus neoformans        consensus sequence consisting essentially of nucleotides        1165-1184 of SEQ ID NO: 2; and    -   (c) instructions for using the forward primer and reverse primer        in performing PCR to detect a presence of Cryptococcus        neoformans in a sample.

Other non-limiting example embodiments may include kits for PCRdetection of Cryptococcus neoformans, which include:

-   -   (a) a forward primer that anneals to a Cryptococcus neoformans        consensus sequence consisting essentially of nucleotides        2105-2124 of SEQ ID NO: 1;    -   (b) a reverse primer that anneals to a Cryptococcus neoformans        consensus sequence consisting essentially of nucleotides 508-527        of SEQ ID NO: 2; and    -   (c) instructions for using the forward primer and reverse primer        in performing PCR to detect a presence of Cryptococcus        neoformans in a sample.

According to non-limiting example embodiments, the kits may furtherinclude at least one hybridization probe having a nucleotide sequenceconsisting essentially of a sequence set forth in SEQ ID NO: 5, whereinthe probe includes at least one fluorescent moiety; and the instructionsmay include instructions for using the forward primer, reverse primerand hybridization probe in performing real-time PCR in detecting apresence of Cryptococcus neoformans in a sample.

Articles of manufacture and kits provided herein may additionallyinclude reagents for carrying out the methods disclosed herein (e.g.,buffers, Taq polymerase enzymes, co-factors, and agents to preventcontamination). Such reagents may be specific for one of thecommercially available instruments described herein.

The following examples are provided to further illustrate variousnon-limiting embodiments and techniques. It should be understood,however, that these examples are meant to be illustrative and do notlimit the scope of the claims. As would be apparent to skilled artisans,many variations and modifications are intended to be encompassed withinthe spirit and scope of the invention.

EXPERIMENTAL EXAMPLES Example 1 PCR Conditions for the Testing ofClinical Samples

(a) Nucleotide Sequences for the Primers and Hybridization Probes

Nucleotide sequences of primer sets and hybridization probes weredetermined from the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO:2, using computer programs of Beacon Designer 4.02 (Build 402003)(PREMIER Biosoft International, Palo Alto, Calif.). Primer sets andhybridization probes having the specified nucleotide sequences werepurchased from Integrated DNA Technologies (Stokie, Ill.). The primerset includes the first forward primer (Cneo F1), and the first reverseprimer (Cneo R1) (See, details in FIG. 1).

(b) PCR Conditions

For the testing of clinical samples, each PCR was conducted in a volumeof 25 μl containing the following: DNA extracted from a clinical sample(e.g., 500 ng), 600 nM of a forward primer, which has the nucleotidesequence set forth in SEQ ID NO:3 (“the Cneo F1”), 600 nM of a reverseprimer, which has the nucleotide sequence set forth in SEQ ID NO: 4(“the Cneo R1”), 200 nM of a hybridization probe, which has thenucleotide sequence set forth in SEQ ID NO: 5 (“the Cneo Probe”), and1×MDL Custom qPCR SuperMix (Quanta BioSciences, Inc., Gaithersburg,Md.). The hybridization probe (“the Cneo Probe”) has a6-carboxy-fluorescein moiety and a Black Hole Quencher 1 moiety attachedto its 5′-end and 3′-end, respective. 1×MDL Custom qPCR SuperMix has aQuanta BioSciences catalog number of 172-5008, and the 2× stock solutionof the SuperMix contained 50 U/ml of AccuStart™ Taq DNA polymerase, 40mM Tris-HCl (pH 8.4), 100 mM KCl, 6 mM MgCl₂, 400 nM dATP, 400 nM dCTP,400 nM dGTP, 800 nM dUTP, 40 U/ml of UNG, and proprietary stabilizers ofQuanta BioSciences, Inc. The Cneo probe was present in the reactionmixture to monitor real-time synthesis of the amplicon resulting fromeach successful PCR. The Cneo F1 primer, the Cneo R1 primer, and theCneo Probe were used at 600 nM and 200 nM, respectively.

(c) Real-Time PCR

Real-Time PCR was conducted using the Rotor-Gene 3000 platform (CorbettResearch, Sydney, Australia). Parameters for each PCR were as follows:an initial incubation at 50° C. for 2 minutes to activate UNG, followedby incubation at 94° C. for 3 minutes to initially denature the DNA,inactivate the UNG, and activate the AccuStart™ Taq DNA polymerase.Next, 35 cycles of denaturation (95° C. for 15 seconds), and anannealing and extension (65° C. for 60 seconds) were performed withfluorescence acquisition (excitation at 470 nM and emission at 510 nM)immediately following each annealing and extension step. Fluorescencecurves were analyzed with dynamic-tube normalization, slope correction,and automatic threshold determination by a best-fit line of threeconcentrations of the positive-control standard DNA using CorbettRobotics Rotor Gene software version 6.0.31 (Corbett Research, Sydney,Australia). The three concentrations of the positive-control standardDNA were 1×10³, 1×10⁵, and 1×10⁷ copies per reaction of pCneoJE (aplasmid which is described in Example 2). Negative template control(NTC) refers to plasmid lacking a template DNA and NTC threshold was setto a maximum 10%. The amplification target for Cryptococcus neoformansfungal specific transcription factor on chromosome 9, and successfulamplification would result in a 129 bp amplicon product.

Example 2 Positive-Control Standard DNA for the PCR

A positive-control standard DNA for the PCR used was prepared fromCryptococcus neoformans (commercially available, ATCC® No. 22344). PCRamplification was conducted using 200 ng of DNA extracted Cryptococcusneoformans. Parameters for this PCR were as follows: an initialincubation at 94° C. for 3 minutes, followed by 40 cycles of incubationat 94° C. for 1 minute, 65° C. for 1 minute, and 72° C. for 2 minutes,followed by a final incubation at 72° C. for 10 minutes. All otherconditions for this reaction were similar to those described inExample 1. The resulting amplicon was cloned into the pCR® 2.1-TOPO®vector (Invitrogen, Carlsbad, Calif.) to produce pCneoJE, which was usedas the positive-control standard DNA.

Different dilutions of pCneoJE plasmid were prepared and the range ofdilution includes 1×10⁹, 1×10⁸, 1×10⁷, 1×10⁶, 1×10⁵, 1×10⁴, 1×10³, and1×10³ copies/dilution. The pCneoJE dilutions were run in a real-timePCR. As shown in FIG. 2, real-time PCR detects fluorescence at a rangeof concentrations ranging from 1×10⁹ to 1×10² copies/dilutions,indicating that the primer set and PCR conditions are optimal indetecting Cryptococcus neoformans. After 36 PCR cycles, the assayreproducibly detects as few as 100 copies/reaction. (See, FIG. 2). Thedata indicate this real-time PCR in the detection of Cryptococcusneoformans is highly sensitive.

Example 3 Specificity of the PCR Using Primers Directed to the FungalSpecific Transcription Factor Gene in Cryptococcus neoformans

The specificity of the PCR utilizing the combination of the Cneo Fprimer, the Cneo R primer, and the Cneo probe was assessed by attemptingto conduct real-time PCRs at varying dilutions of extracted DNA fromCryptococcus neoformans obtained from ATCC source (ATCC® No. 2344). Asshown in FIG. 3, real-time PCR detects positive reactivity towardsCryptococcus neoformans at dilutions of 1:10, 1:100, 1:1,000.

We next tested forty-seven (47) species listed in the 10 cocktailformats. Duplicate reaction mixtures were tested and each reactionmixture contained DNA extracted from four or five types of pathogensummarized in Table 1. Each pathogen was purchased from ATCC®, exceptfor Candida lusitaneae which was purchased from MicroBioLogics Inc.(Saint Cloud, Minn.).

TABLE 1 Number Cocktail 1 Gardnerella vaginalis ATCC ® No. 14018Neisseria gonorrhoeae ATCC ® No. 27628 Trichomonas vaginalis ATCC ® No.30246 Ureaplasma urealyticum ATCC ® No. 27618 Chlamydia trachomatisATCC ® No. VR-901B Cocktail 2 Bacteroides fragilis ATCC ® No. 23745Mobiluncus curtisii ATCC ® No. 35241 Mobiluncus mulieris ATCC ® No.35243 HTLV-I ATCC ® No. CRL-8294 Human herpesvirus 6B ATCC ® No. VR-1467Cocktail 3 Herpes simplex virus 1 ATCC ® No. VR-539 Herpes simplex virus2 ATCC ® No. VR-734 Human Papillomavirus ATCC ® No. CRL-1550Epstein-Barr virus ATCC ® No. CCL-86 Cytomegalovirus ATCC ® No. VR-807Cocktail 4 Candida albicans ATCC ® No. 11651 Candida glabrata ATCC ® No.2001 Candida parapsilosis ATCC ® No. 22019 Candida tropicalis ATCC ® No.13803 Aspergillus fumigatus ATCC ® No. 14110 Cocktail 5 Mycoplasmafermentans ATCC ® No. 15474 Mycoplasma pneumoniae ATCC ® No. 15377Mycoplasma genitalium ATCC ® No. 33530 Mycoplasma penetrans ATCC ® No.55252 Mycoplasma hominis ATCC ® No. 14027 Cocktail 6 Human herpesvirus-8ATCC ® No. CRL-2230 Adenovirus type 1 ATCC ® No. VR-1 CoxsackievirusATCC ® No. VR-184 Babesia microti ATCC ® No. 30222 Cocktail 7 Chlamydiapneumoniae ATCC ® No. VR-1356 Helicobacter pylori ATCC ® No. 43579Brucella ovis ATCC ® No. 25840 Borrelia burgdorferi ATCC ® No. 35210Canine herpesvirus ATCC ® No. VR-552 Cocktail 8 Bartonella henselaeATCC ® No. 49882 Bartonella bacilliformis ATCC ® No. 35686 BartonellaQuintana ATCC ® No. 51694 Trichosporon cutaneum ATCC ® No. 4151 Cocktail9 Influenza virus A ATCC ® No. VR-1520 Haemophilus parainfluenzae ATCC ®No. 7901 Human rhinovirus 6 ATCC ® No. VR-1116AS/GP Human rhinovirus 11ATCC ® No. VR-1121 Adenovirus type 10 ATCC ® No. VR-11 Cocktail 10Candida krusei ATCC ® No. 14243 Candida lusitaniae MicroBioLogics No.0774P Candida dubliniensis ATCC ® No. MYA-179 Candida utilis ATCC ® No.9226

FIG. 3 depicts an exemplary quantitation data display in real-time PCR.While we observed amplification with various dilutions of extracted DNAof Cryptococcus neoformans, no amplification is seen in the eightcocktails tested (i.e., cocktails 1-10) (i.e., fluorescence below Ctthreshold; see FIG. 3). The contents of each pathogen cocktail arelisted in Table 1.

Further shown in the following Table 2, no PCR amplification wasobserved in any of the cocktails tested (i.e., cocktails 1-10). In thisseries of studies, two samples were run separately. Neither of the tworeaction mixtures containing DNA from Cocktails 1 through 10 waspositive when using the combination of the Cneo F1 primer, Cneo R1primer and the Cneo Probe (see Table 2). These data support that theprimer probes and hybridization probe provide a high degree ofspecificity and accurate detection of Cryptococcus neoformans. No falsepositives were observed, among the 47 organisms tested.

TABLE 2 Samples Results Cocktail 1 - Sample 1 −* Cocktail 1 - Sample 2 −Cocktail 2 - Sample 1 − Cocktail 2 - Sample 2 − Cocktail 3 - Sample 1 −Cocktail 3 - Sample 2 − Cocktail 4 - Sample 1 − Cocktail 4 - Sample 2 −Cocktail 5 - Sample 1 − Cocktail 5 - Sample 2 − Cocktail 6 - Sample 1 −Cocktail 6 - Sample 2 − Cocktail 7 - Sample 1 − Cocktail 7 - Sample 2 −Cocktail 8 - Sample 1 − Cocktail 8 - Sample 2 − Cocktail 9 - Sample 1 −Cocktail 9 - Sample 2 − Cocktail 10 - Sample 1 − Cocktail 10 - Sample 2− Negative Control (no template DNA) − Positive Control (genomic DNApurified from +** Cryptococcus neoformans ATCC ® No. 2344 at 1:10dilution) Positive Control (genomic DNA purified from + Cryptococcusneoformans ATCC ® No. 2344 at 1:100 dilution) Positive Control (genomicDNA purified from + Cryptococcus neoformans ATCC ® No. 2344 at 1:1,000dilution) *“−” indicates the absence of PCR amplification in the sample.**“+” indicates the presence of PCR amplification in the sample.

Example 4 Precision of the PCR Using Primers Directed to the FungalSpecific Transcription Factor in Cryptococcus neoformans

To determine the precision of the PCR when using primers directedspecifically to the fungal specific transcription factor in Cryptococcusneoformans, five (5) technicians were asked to independently conduct thereal-time PCR. The study was performed in double-blinded manner, thatis, none of the technicians knew the identity of the template DNA in thereaction mixture before and while they were conducting the PCRs.

Each of five (5) technicians independently assessed the precision of thePCR utilizing the combination of the Cneo F1 primer, the Cneo R1 primer,and the Cneo probe by attempting to conduct real-time PCRs using DNAobtained from urine samples, wherein each of nine reaction mixtures wasknown to be free of DNA of Cryptococcus neoformans, and each of ninereaction mixtures was spiked with DNA of Cryptococcus neoformans ATCC®No. 2344.

As summarized in Table 3, all five technicians correctly determinedwhich of the eighteen reaction mixtures contained DNA of Cryptococcusneoformans and which did not, even though they did not know the identityof the template DNA in each reaction mixture before attempting toconduct the PCRs.

TABLE 3 Technician Sample Expected A B C D E 1  −* − − − − − 2 − − − − −− 3 − − − − − − 4 − − − − − − 5 − − − − − − 6 − − − − − − 7 − − − − − −8 − − − − − − 9 − − − − − − 10   +** + + + + + 11 + + + + + +12 + + + + + + 13 + + + + + + 14 + + + + + + 15 + + + + + +16 + + + + + + 17 + + + + + + 18 + + + + + + Negative Control − − − − −− (no template DNA) Positive Control + + + + + + (1 × 10³ copies ofpCneoJE) Positive Control + + + + + + (1 × 10⁵ copies of pCneoJE)Positive Control + + + + + + (1 × 10⁷ copies of pCneoJE) *“−” indicatesthe absence of DNA of Cryptococcus neoformans in the sample. **“+”indicates the presence of DNA of Cryptococcus neoformans in the sample.

Example 5 PCR Using Two (2) Different Set of Primers Directed to theSame Fungal Specific Transcription Factor Gene in Cryptococcusneoformans

(a) Nucleotide Sequences for Two (2) Additional Sets of PCR Primers

To further illustrate that primers directed against the fungal specifictranscription factor gene is specific for Cryptococcus neoformans in aconventional PCR reaction, two (2) additional primer sets (e.g., forwardprimer, and reverse primer) directed against the same gene inCryptococcus neoformans was prepared. We designed the additional primersets for the fungal specific transcription factor gene in Cryptococcusneoformans. The forward and reverse primers were designed using BeaconDesigner 4.02 (Build 402003) (PREMIER Biosoft International, Palo Alto,Calif.). The primer sets having the specified nucleotide sequences werepurchased from Integrated DNA Technologies (Stokie, Ill.).

The second primer set contains a second forward primer (Cneo F2) and asecond reverse primer (Cneo R2), the nucleotide sequence of which is setforth in Table 4. The third primer set contains a third forward primer(Cneo F3) and a third reverse primer (Cneo R3), the nucleotide sequenceof which is set forth in Table 4.

TABLE 4 Amplicon Name Primer sequences Location Size Cneo F25′-TAC AA TCG GTC JEC21 116 bp ACG GTA TT-3′ Chromosome 9(SEQ ID NO.: 6) Cneo R2 5′-CTT CGT CTC ATA CCC AGA TC-3′ (SEQ ID NO.: 7)Cneo F3 5′-GGA TGC TTG GAA JEC21 148 bp GGA TCT AT-3′ Chromosome 9(SEQ ID NO.: 8) Cneo R3 5′-ATG ACC GTC TAT CCA GTG TA-3′ (SEQ ID NO.: 9)

(b) PCR Conditions

Conventional PCR was conducted. In brief, 25 μl containing the extractedDNA (e.g., 500 ng), 600 nM of the second primer set (i.e., Cneo F2 andCneo R2) (SEQ ID NOs: 6 and 7), and 1×MDL Custom qPCR SuperMix (QuantaBioSciences, Inc., Gaithersburg, Md.). 1×MDL Custom qPCR SuperMix has aQuanta BioSciences catalog number of 172-5008, and the 2× stock solutionof the SuperMix contained 50 U/ml of AccuStart™ Taq DNA polymerase, 40mM Tris-HCl (pH 8.4), 100 mM KCl, 6 mM MgCl₂, 400 nM dATP, 400 nM dCTP,400 nM dGTP, 800 nM dUTP, 40 U/ml of UNG, and proprietary stabilizers ofQuanta BioSciences, Inc. The reaction mixture was monitored by gelelectrophoresis (ethidium bromide staining) of the amplicon resultingfrom each successful PCR.

We examined if the second primer set (i.e., the Cneo F2 primer and theCneo R2 primer) provides a successful amplification product forCryptococcus neoformans (neat) and a concentration range of 1:10 and1:100 dilutions of Cryptococcus neoformans (i.e., extract DNA from ATCC2344). (lanes 1-4, FIG. 4).

Similarly, we examined if the third primer set (i.e., the Cneo F3 primerand the Cneo R3 primer) provides a successful amplification product forCryptococcus neoformans (neat) and a concentration range of 1:10 and1:100 dilutions of Cryptococcus neoformans (i.e., extract DNA from ATCC2344). (lanes 6-9, FIG. 4).

As shown in FIG. 4, both the second primer set and the third primer setproduce an amplicon products (size of 116 bp and 148 bp, respectively)(see lanes 1 and 6, FIG. 4). This result indicates that the fungalspecific transcription factor gene is specific for Cryptococcusneoformans.

Based on these data, it is concluded that primer sets directed againstthe fungal specific transcription factor gene of Cryptococcus neoformansprovide a specific PCR tool to detect Cryptococcus neoformans. Theprimers provide specific amplification of an amplicon product forCryptococcus neoformans and not other related species.

Example 6 Percent Identity (% Sequence Identity) Studies

One skilled in the art would appreciate that about 99%, 95%, and 90%sequence identity would behave the same as that of 100% with respect tothe ability to anneal to a target DNA and initiate a PCR reaction. Inthis study, we set out to determine if 80% or less sequence identity maybehave as that of 100%. A primer set against the fungal specifictranscription factor gene of Cryptococcus neoformans was synthesized,which has 80% identity to SEQ ID NOs: 3 and 4 (i.e., the forward primeris 80% identical to SEQ ID NO: 3 and the reverse primer is 80% identicalto SEQ ID NO: 4, respectively). (see Table 5).

TABLE 5 Primers Sequences PCR Results 100% Identity 5′-GAC ATC GAT CTG+* to Cneo F1 CCA TAC TCA TCG-3′ (SEQ ID NO.: 3) 100% Identity5′-GCG TCA CAC TAC to Cneo R1 AGG TCA GTT G-3′ (SEQ ID NO.: 4)80% Identity 5′-GAC ATC GAT CTG −** to Cneo F1 CCA TAC T-3′(i.e., Cneo F4) (SEQ ID NO.: 10) 80% Identity 5′-GCG TCA CAC TACto Cneo R1 AGG TC-3′ (i.e., Cneo R4) (SEQ ID NO.: 11) *“+” indicates thea presence of PCR amplification in the sample **“−” indicates theabsence of PCR amplification in the sample.

As shown in Table 5, the primer set (i.e., Cneo F4 and Cneo R4) having80% sequence identity to the first primer set (SEQ ID NOs. 3 and 4) didnot provide any PCR amplicon, indicating that the 80% identity primerset is not sufficient to anneal to the template nucleic acid during thereal-time PCR, and thus account for the absence of PCR reaction. Thisresult suggests that at least greater than 80% sequence identity isrequired.

Example 7 Lack of Specificity of the PCR: Using Primers Against TwoAnother Genes in Cryptococcus neoformans

To illustrate the specificity of the fungal specific transcriptionfactor gene in the PCR reaction, we further designed primer setsdirected against two different genes. One primer set is againstCryptococcus neoformans var. neoformans JEC21 chromosome 2, the fulllength nucleotide sequence is set forth in GeneBank Accession No.AE017342. Another primer set is against Cryptococcus neoformans var.grubii strain H99 anti-phagocytic protein 1, the full length nucleotidesequence is set forth in GeneBank Accession No. AY965856.

The Cryptococcus neoformans var. neoformans JEC21 chromosome 2 contains1,632,307 nucleotides. The Cryptococcus neoformans var. grubii strainH99 anti-phagocytic protein 1 gene contains 574 nucleotides.

In one study, the primer set was against the Cryptococcus neoformansJEC21 chromosome 2 and has the nucleotide sequences as set forth inTable 6 (i.e., SEQ ID NOs: 12, 13 and 14). The primer set wassynthesized and used in a real-time PCR with conditions described inExample 1.

In another study, the primer set was against the Cryptococcus neoformansgrubii strain H99 anti-phagocytic protein and has the nucleotidesequences as set forth in Table 6 (i.e., SEQ ID NOs: 15, 16 and 17). Theprimer set was synthesized and also used in a real-time PCR withconditions described in Example 1.

TABLE 6 Base Name Gene Location Pair Sequences Cneo F5 Cryptococcus116 bp 5′-CGT TAT GAA GCT TCA AAT neoformans var. CC-3′ (SEQ ID NO.: 12)Cneo R5 neoformans JEC21 5′- TTA CTC GTT CAG TCA TAT Chromosome 2 ATG-3′(SEQ ID NO.: 13) Cneo Probe  5′/56-FAM/AGG CGA TAG CAG 5AAC CAC AAC /3BHQ_1/-3′ (SEQ ID NO.: 14) Cneo F6 Cryptococcus 131 bp5′-CAA CCC AAC AAA TAA TAC neoformans var. ATG-3′ (SEQ ID NO.: 15)Cneo R6 grubii strain H99 5′-GTT AGC CTT TCT AAG ATC TC- anti-phagocytic3′ (SEQ ID NO.: 16) Cneo Probe  protein 1 5′/56-FAM/ TCC TCT GCC ACT GCT6 GAA CT/3BHQ_1/ (SEQ ID NO.: 17)

As shown in the following Table 7, the forward primer (Cneo F1), reverseprimer (Cneo R1) and hybridization probe (Cneo probe) successfullydetect the presence of Cryptococcus neoformans. This serves as thepositive control and illustrates that the real-time PCR system wasadequate and operational. Fluorescence detection goes as low as1:100,000 dilutions, indicating that the PCR assay is highly sensitive.

However, the primer set #5 (i.e., Cneo F5, Cneo R5 and Cneo Probe 5)failed to detect the presence of Cryptococcus neoformans in real-timePCR, at the tested dilution ranges of 1:10 to 1:10,000. (see Table 7).Similarly, the primer set #6 (i.e., Cneo F6, Cneo R6 and Cneo Probe 6)also failed to detect the Cryptococcus neoformans. Unlike the fungalspecific transcription factor gene, prime sets against two other genes(i.e., Cryptococcus neoformans var. neoformans JEC21 chromosome 2 andCryptococcus neoformans var. grubii strain H99 anti-phagocyticprotein 1) both fail to serve as specific markers for Cryptococcusneoformans.

TABLE 7 Sample Nos. Samples Results Forward Primer: Cneo F5 (SEQ ID NO.:12) Reverse Primer: Cneo R5 (SEQ ID NO.: 13) Hybridization Probe: CneoProbe 5 (SEQ ID NO.: 14) 1 C. neoformans (1:10) −** 2 C. neoformans(1:100) − 3 C. neoformans (1:1,000) − 4 C. neoformans (1:10,000) − 5 NTC− 6 NTC − Forward Primer: Cneo F6 (SEQ ID NO.: 15) Reverse Primer: CneoR6 (SEQ ID NO.: 16) Hybridization Probe: Cneo Probe 6 (SEQ ID NO.: 17) 7C. neoformans (1:10) − 8 C. neoformans (1:100) − 9 C. neoformans(1:1,000) − 10 C. neoformans (1:10,000) − 11 NTC − 12 NTC − ForwardPrimer: Cneo F1 (SEQ ID NO.: 3) Reverse Primer: Cneo R1 (SEQ ID NO.: 4)Hybridization Probe: Cneo Probe (SEQ ID NO.: 5) 13 C. neoformans (1:10)+* 14 C. neoformans (1:100) + 15 C. neoformans (1:1,000) + 16 C.neoformans (1:10,000) + 17 NTC − 18 NTC − NTC: negative template control(i.e., no DNA template) *“+” indicates the presence of PCR amplificationin the sample. **“−” indicates the absence of PCR amplification in thesample.

FIG. 5 depicts fluorescence emitted in an exemplary real-time PCRreaction. Positive control primer set #1 (i.e., Cneo F1, Cneo R1, andCneo probe) was used. Dilution of 1:10, 1:100, and 1:1,000 of extractedDNA of ATCC® 2344 was used. While the primer set #1 was able to detectthe presence of Cryptococcus neoformans, both the primer sets #5 and 6failed to do so (i.e., fluorescence was below the threshold). (FIG. 5).

Although the invention has been described in example embodiments,additional modifications and variations would be apparent to thoseskilled in the art. It is therefore to be understood that the inventionsherein may be practiced other than as specifically described. Thus, thepresent embodiments should be considered in all respects as illustrativeand not restrictive. Accordingly, it is intended that such changes andmodifications fall within the scope of the present invention as definedby the claims appended hereto.

1. An isolated hybridization probe, wherein said probe has a nucleotidesequence consisting essentially of SEQ ID NO:
 5. 2. The isolatedhybridization probe of claim 1, wherein said probe comprises afluorescent moiety.
 3. The isolated hybridization probe of claim 2,wherein said fluorescent moiety is 6-carboxy-fluorescein.
 4. A kit forPCR detection of Cryptococcus neoformans, comprising the isolatedhybridization probe of claim 1, said kit further comprises: (a) aforward primer that anneals to a Cryptococcus neoformans consensussequence consisting essentially of nucleotides 1121-1144 of SEQ ID NO:1; (b) a reverse primer that anneals to a Cryptococcus neoformansconsensus sequence consisting essentially of nucleotides 1512-1533 ofSEQ ID NO: 2; and (c) an instruction for using said forward primer andreverse primer in performing PCR to detect the presence of Cryptococcusneoformans in a sample.
 5. The kit of claim 3, wherein said forwardprimer has a nucleotide sequence consisting essentially of SEQ ID NO: 3and said reverse primer has a nucleotide sequence consisting essentiallyof SEQ ID NO:
 4. 6. A kit for PCR detection of Cryptococcus neoformans,comprising the isolated hybridization probe of claim 1, said kit furthercomprises: (a) a forward primer that anneals to a Cryptococcusneoformans consensus sequence consisting essentially of nucleotides1480-1499 of SEQ ID NO: 1; (b) a reverse primer that anneals to aCryptococcus neoformans consensus sequence consisting essentially ofnucleotides 1165-1184 of SEQ ID NO: 2; and (c) an instruction for usingsaid forward primer and reverse primer in performing PCR to detect thepresence of Cryptococcus neoformans in a sample.
 7. The kit of claim 5,wherein said forward primer has a nucleotide sequence consistingessentially of SEQ ID NO: 6 and said reverse primer has a nucleotidesequence consisting essentially of SEQ ID NO:
 7. 8. A kit for PCRdetection of Cryptococcus neoformans, comprising the isolatedhybridization probe of claim 1, said kit further comprises: (a) aforward primer that anneals to a Cryptococcus neoformans consensussequence consisting essentially of nucleotides 2105-2124 of SEQ ID NO:1; (b) a reverse primer that anneals to a Cryptococcus neoformansconsensus sequence consisting essentially of nucleotides 508-527 of SEQID NO: 2; and (c) an instruction for using said forward primer andreverse primer in performing PCR to detect the presence of Cryptococcusneoformans in a sample.
 9. The kit of claim 7, wherein said forwardprimer has a nucleotide sequence consisting essentially of SEQ ID NO: 8and said reverse primer has a nucleotide sequence consisting essentiallyof SEQ ID NO: 9.